Our work focuses on the use of DNA-based vaccine strategies both as preventive and immunotherapeutic approaches. We have generated efficient SIV and HIV DNA expression vectors. This work is based on our previous recognition that RNA elements (called INS) present within the gag/pol and env coding regions of HIV are responsible for nuclear retention and instability of the transcripts in the absence of Rev, and that these elements can be eliminated by changing the nucleotide composition of the transcripts (RNA optimization;also referred to as codon optimization) without affecting the amino acid sequence. The immunogencity of the antigens was further improved by modifying the trafficking of the antigens. The introduced modifications of the proteins led to more efficient secretion of the SIV antigens resulting in increased cellular and humoral immune responses in the vaccinated mice or rhesus macaques. Using DNA-only vaccination, we found that our optimized DNA vaccine vectors are able to induce potent immune responses able to protect from high viremia in the rhesus macaque/SIVmac251 challenge model. A limitation in using DNA as a vaccine is its relative inefficient delivery upon intramuscular injection. Recent developments to improve DNA delivery include in vivo electroporation. We recently reported that electroporation dramatically increased the efficiency of DNA delivery in both SIV experienced and nave rhesus macaques, leading to greatly augmented antigen expression. We found that this vaccination method results in enhanced immune responses with a high frequency of circulating SIV-specific T cells, the presence of multifunctional T cells, and induction of both effector memory and central memory CD4 and CD8 SIV-specific T cells. In addition to systemic immune responses, the use of this improved DNA vaccination methodology also induced mucosal cellular and humoral SIV-specific responses. Importantly, we found that immune responses were also long-lasting and could be detected more than 2 years after the last vaccination. Challenge of animals which received this optimized vaccination regimen showed a significant improvement in virological control of the highly pathogenic SIVmac251 challenge. Thus, efficient DNA delivery methods in combination with improved DNA vaccine cocktails resulted in greatly augmented and more balanced immune responses in vaccinated rhesus macaques. This will allow us to further examine and dissect immune responses in different tissues and to correlate immunological parameters to virus control efficacy. The study of antiretroviral (ART)-treated SIV-infected animals provides measurements of recall responses and the capability to test the efficacy of the vaccination approach upon release from ART. Strategies to restore immune function that lead to better control of viral replication promise to be beneficial for the clinical management of HIV-infected individuals. We found that DNA-only vaccination during ART was able to evoke very high levels of SIV-specific recall immune responses that led to significant, long-lasting virological benefit in 60% of animals after release from ART. In a recent follow-up study, we found that repeated DNA therapeutic vaccination of chronically SIV-infected macaques provides an additional persistent virological benefit (1 log drop in viremia), which supports the concept that therapeutic vaccination together with HAART regimen could be beneficial to HIV-infected persons. On the basis of these results, we propose that to test the efficacy of DNA-only vaccination in mounting recall responses in HAART-treated people. We also focused on further optimizing the DNA-based vaccination approach by modifying the vaccination cocktail. The presence of the cytokine DNAs (IL-12 and IL-15) was found to improve the quantity and alter the quality of the immune responses. To optimally use these cytokines, we studied their regulation and found that IL-15/IL-15Ra as well as the IL-12 cytokine family use similar posttranscriptional and posttranslational regulation. As a result, the formation and secretion of the subunits and heterodimers are highly regulated steps. Using this knowledge, we have generated optimized expression vectors, which allow their efficient use in animal models, and which could also be important for future translational applications. To design better vaccine strategies, it is important to dissect correlates of protective immunity. Among the 'controllers'are animals from our vaccine studies as well as animals infected with Rev-independent live-attenuated SIV strains (LASIV), which serve as model to study the underlying mechanisms of protection from disease development. Such animals allow us to dissect the cellular and viral determinants that contribute to disease development and they also provide a unique resource to study mechanisms leading to protective immunity. To analyze the immune mechanisms responsible for viral control, 5 macaques infected at day 1 after birth were subjected to CD8+ cell depletion at 6.7 years after infection. This resulted in viremia increases to 3.7-5.5 log10 RNA copies, supporting a role of CD8-mediated responses in the control of viral replication. The rebounding viremia was rapidly controlled to levels below the threshold of detection, and occurred in the absence of SIV-specific CD8+ T cells and significant CD8+ T cell recovery in 4 of the 5 animals, suggesting that other mechanisms are involved in the immunological control of viremia. Monitoring immune responses at the time of viral control demonstrated a burst of circulating SIV-specific CD4+ T cells characterized as CD45RA-CD28+CD95+CCR7- and also Granzyme B+, suggesting cytotoxic ability. Control of viremia was also concomitant with increases in humoral responses to Gag and Env, including a transient increase in neutralizing antibodies against the neutralization-resistant SIVmac239 in 4 of 5 animals. These data demonstrate that a combination of cellular responses mediated by CD4+ T cells and humoral responses were associated with the rapid control of the rebounding viremia in macaques infected by the Rev-independent live-attenuated SIV, even in the absence of measurable SIV-specific CD8+ T cells in the blood, emphasizing the importance of different components of the immune response for full control of SIV infection. These studies will provide critical information about the establishment and maintenance of host immune responses during chronic retroviral infections with distinct pathogenic outcomes and will facilitate further improvements of DNA vaccines.